Protocol for tissue clearing and 3D analysis of dopamine neurons in the developing mouse midbrain

Advances in tissue clearing enable analysis of complex migratory patterns of developing neurons in whole intact tissue. Here, we implemented a modified version of 3DISCO to study migration of midbrain dopamine (DA) neurons. We provide a detailed protocol starting from whole-brain immunostaining, tissue clearing, and ultramicroscopic imaging to post-acquisition quantification and analysis. This protocol enables precise quantification of DA neuron migration but can also be applied more generally for analyzing neuron migration throughout the nervous system. For complete details on the use and execution of this protocol, please refer to Brignani et al. (2020). [Display omitted] •3D imaging of cleared brains enables analysis of complex neuronal migratory patterns•3D visualization of dopamine neuron subsets using clearing and lightsheet imaging•Detailed procedure for the analysis of neuronal subset positioning in the DA system•Detailed methods to analyze neuronal migratory patterns during brain development Advances in tissue clearing enable analysis of complex migratory patterns of developing neurons in whole intact tissue. Here, we implemented a modified version of 3DISCO to study migration of midbrain dopamine (DA) neurons. We provide a detailed protocol starting from whole-brain immunostaining, tissue clearing, and ultramicroscopic imaging to post-acquisition quantification and analysis. This protocol enables precise quantification of DA neuron migration but can also be applied more generally for analyzing neuron migration throughout the nervous system.